Background: Use and knowledge of medicinal plants play an essential role in community health in rural Mexico. They are part of the local inheritance and constitute an economic alternative. Nevertheless, knowledge of their use has declined due to factors like accelerated urbanization processes. Some authors have proposed that by reducing natural spaces, urbanization generates changes which will impact recognition, use and management of natural resources. We evaluate how urbanization affects knowledge, use and perception of medicinal plants in a Biosphere Reserve in Mexico. Methods: Using a mixed methodology including quantitative and qualitative analyses, we generate a list of medicinal plants, methods of preparation, prevalence of illness, and use. Results: 210 medicinal plants were identified. The more urbanized community used greater number of introduced plants; whereas the less urbanized used and had more knowledge about wild plants. One of the factors explaining these differences was occupation. People who work outdoors have greater knowledge of wild plants. The more urbanized community knew and used more introduced species. Conclusions: This can lead to a loss of knowledge of use and management of wild species, with implications for the conservation of biocultural heritage. Substitution of native by introduced plants shows disinterest and disuse, which could be reflected in their ecosystems.